Femto3D Atlas

Overview
The flexible movability of the focal point established by the acousto-optic scanner and the measurement features of the control software enable the user to perform imaging along numerous patterns. These patterns have been developed based on scientific requests and aimed to support various neuroscientific applications such as neural network and dendritic imaging. The deep tissue penetration of the laser, the near cubic meter scanning volume, the submicron spatial resolution and the high scanning speed enable the researchers to follow intra- and intercellular processes in living brain or any other organs.

Scanning modes

3D random-access point scanning

3D random-access point scanning is the fastest method available with two-photon microscopes to read-out neuronal activity. It enables multiple points, distributed in 3D, to be imaged simultaneously. Within a large scanning volume, it can reach the 30 kHz scanning speed which is approximately one million times faster than other frame-by-frame scanning methods. This imaging speed means that thousands of individual neurons (e.g. in different cortical layers) can be measured with microsecond resolution simultaneously, revealing – for example – the dynamics of neuronal networks.

3D trajectory scanning

3D trajectory scanning has been improved from 3D random-access point scanning. The points are extended by drifting the focal point along short 3D trajectories which allows imaging without interruption at multiple dendritic branches. The sampling is continuous during the drift, so this scanning mode gives a more detailed spatial resolution without changing the overall scanning time: this remains as high as during the point scanning. As a result, the function of thin dendritic segments, or even spines and single action potentials can be revealed.

Anti-motion technology

For motion correction

Our Anti-motion technology is an acousto-optic scanner-based imaging method using drift scanning technology which has been successfully improved for correcting motions that appear during behavior. To cover that area where fluorescent signals have occurred due to motion, random-access points are extended to drifting lines. The lines can be precisely fitted to each other, resulting in surface or 3D volume elements as enlarged areas. These elements cover not only the pre-selected ROIs but also the neighboring areas making it possible to preserve all information provided by fluorescence during brain motion. This allows motion artifact and neuropil contamination to be eliminated by computer algorithms. The implemented motion artifact elimination algorithm has been shown to increase the SNR by more than one order of magnitude in behaving animals. See also Szalay et al., Neuron, 2016.

3D Chessboard scanning
Chessboard scanning is a planar extension of random-access point scanning, which extends scanning points localized in 3D to small squares by drifting the laser beam. The name, chessboard, is derived from the layout, which is generated by arranging all the squares side-by-side to get a chessboard like pattern containing the selected regions with somata and the surrounding areas. This pattern allows visualization of the somata activity, handling and storing of the data and, importantly, correcting for motion to be carried out simultaneously.

3D Multi-layer, multi-frame scanning
Imaging of multiple frames with different sizes and at any position in the scanning volume can be used to follow all events propagating along the cell. The figure shows imaging of the entire length of a pyramidal neuron, where the small scanned rectangles covering the areas around the cell enable us to record fluorescent signals and responses to visual stimuli.

3D Multi-cube scanning
Multi-cube scanning is a spatially extended mode of chessboard scanning, where a Z dimension is added to the scanning squares to cover the entire volume of the somata. In this way, fluorescence information from somata is better preserved during larger motions.

3D Multiple-line scanning
This method is based on the 3D random-access point scanning and similar to the 3D trajectory scanning. It enables a huge number of short lines to be measured near simultaneously, therefore it can be used for imaging hundreds of spines. The direction of the lines is set to be parallel to the average motion of the brain, which helps to maximally preserve fluorescence information to eliminate any motion artefacts, so this method is suitable for imaging in awake, behaving animals.

3D Ribbon scanning
Ribbon scanning is an area scanning related extension of the 3D multiple-line scanning mode performed by Anti-motion technology. The neighboring area around the trajectory is also captured by generating drifts either parallel or orthogonal to the trajectory. In this way, it is possible to follow the 3D curvature of one or more dendrites with their spines at the same time retaining fluorescent information during motion.

3D Snake scanning
3D Snake scanning is a volume scanning related extension of ribbon scanning and contains the entire 3D environment of the trajectory. It therefore supports imaging of entire dendritic segments during defined surgical or behavioral protocols, even when the amplitude of motion is very large.

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